A genetic screen identifies C. elegans eif-3.H and hrpr-1 as pro-apoptotic genes and potential activators of egl-1 expression

During C. elegans development, 1090 somatic cells are generated of which 131 reproducibly die, many through apoptosis. The C. elegans BH3-only gene egl-1 is the key activator of apoptosis in somatic tissues, and it is predominantly expressed in ‘cell death' lineages i.e. lineages in which apoptotic cell death occurs. egl-1 expression is regulated at the transcriptional and post-transcriptional level. For example, we previously showed that the miR-35 and miR-58 families of miRNAs repress egl-1 expression in mothers of ‘unwanted' cells by binding to the 3′ UTR of egl-1 mRNA, thereby increasing egl-1 mRNA turnover. In a screen for RNA-binding proteins with a role in the post-transcriptional control of egl-1 expression, we identified EIF-3.H (ortholog of human eIF3H) and HRPR-1 (ortholog human hnRNP R/Q) as potential activators of egl-1 expression. In addition, we demonstrate that the knockdown of the eif-3.H or hrpr-1 gene by RNA-mediated interference (RNAi) results in the inappropriate survival of unwanted cells during C. elegans development. Our study provides novel insight into how egl-1 expression is controlled to cause the reproducible pattern of cell death observed during C. elegans development.


Description
Programmed cell death removes unwanted cells and helps shape organs during development (Suzanne and Steller, 2013).Dysregulation of programmed cell death contributes to several diseases such as cancer, neurodegenerative or autoimmune diseases (Favaloro et al., 2012).Caenorhabditis elegans (C.elegans) is a powerful model for studying programmed cell death.Programmed cell death during C. elegans development occurs in a highly reproducible pattern.Sulston and co-workers discovered that, among 1090 somatic cells generated during the development of a C. elegans hermaphrodite, precisely 131 cells die, many through apoptosis (Conradt et al., 2016;Horvitz, 1999;Sulston and Horvitz, 1977;Sulston et al., 1983).The process of apoptosis is tightly regulated through a genetic pathway that is evolutionarily conserved from nematodes to mammals.In C. elegans, this pathway consists of four key components: egl-1, ced-9, ced-4, and ced-3 (Conradt et al., 2016;Horvitz, 1999).The egl-1 gene is necessary and sufficient for apoptosis and encodes a pro-apoptotic BH3-only protein, EGL-1, which binds to the anti-apoptotic Bcl-2-like protein CED-9 in unwanted cells.This displaces a dimer of the Apaf1-like protein CED-4 from CED-9, thereby allowing CED-4 to form the apoptosome, which facilitates the autocatalytic activation of the CED-3 caspase.Activated CED-3 cleaves multiple substrates, ultimately leading to cell death.In contrast to ced-9, ced-4, and ced-3, which appear to be broadly expressed at least during C. elegans embryogenesis (Chen et al., 2000;Maurer et al., 2007), egl-1 expression is essentially restricted to cell death lineages (Conradt and Horvitz, 1999;Nehme et al., 2010).Thus, the spatiotemporal pattern of egl-1 expression and, hence, the control of egl-1 expression is critical for the highly reproducible pattern of cell death observed during C. elegans development.
egl-1 expression during C. elegans development is regulated at the transcriptional level by lineage-specific transcription factors that act through specific cis-acting elements upstream or downstream of the egl-1 transcription unit (Conradt et al., 2016).In addition, egl-1 expression is controlled at the post-transcriptional level by miR-35 and miR-58 family miRNAs that act through the 3′ UTR of the egl-1 mRNA to repress egl-1 expression in mothers of unwanted cells, thereby preventing their precocious death (Sherrard et al., 2017).Apart from binding sites for miR-35 and miR-58 family microRNAs, the egl-1 3' UTR contains additional conserved elements (Extended data figure 1).For this reason, we propose that factors other than microRNAs, such as RNA-binding proteins (RBPs), may contribute to the post-transcriptional regulation of egl-1 expression and, hence, the highly reproducible pattern of cell death during C. elegans development.
To identify RBPs that promote egl-1 expression, we performed a systematic RNAi (RNA-mediated interference) screen in C. elegans.To that end, we first generated a comprehensive list of previously reported C. elegans RBPs.An initial list of C. elegans RBP-encoding genes published by Wang et al. contains 319 genes that were identified by searching for genes encoding RNA-binding domains (RBDs) (Wang et al., 2009).By searching for additional putative RBDs, Tamburino et al. increased the number of putative C. elegans RBP-encoding genes from 319 to 887 (Extended data table 1a) (Tamburino et al., 2013).They included additional putative RBDs and protein classes such as dsRBDs and ribosomal proteins as well as C2H2 zinc finger-and SAM domain-containing proteins.In addition, systematic approaches were employed to experimentally map mRNA-binding proteins in yeast and mammalian cells by capturing in vivo cross-linked mRNA-protein complexes and by identifying associated proteins by mass spectrometry (Scherrer et al., 2010;Tsvetanova et al., 2010).In a poly(A)-containing mRNA-capturing experiment, Matia-González et al. identified 594 proteins that interact with polyadenylated mRNAs in C. elegans (Matia-González et al., 2015).These mRNA-binding proteins are encoded by 591 genes (Extended data table 1b).
However, only a small fraction of these 591 RBP genes (151) overlaps with the 887 RBP genes reported by Tamburino et al. (Extended data figure 2A).In addition, many previously reported RBPs, such as GLD-3 (Eckmann et al., 2002), MEX-3 and PUF-8 (Ariz et al., 2009), are missing from this list of 591 RBP genes, suggesting that the RBPs identified by Matia-González et al. do not represent all RBPs in C. elegans.Thus, we incorporated the lists published by Tamburino et al. and Matia-González et al. and conducted Gene Ontology (GO) and phenotype enrichment analyses (Extended data figure 2B, Extended data table 1c-1e).Genes with general functions, such as genes encoding tRNA-binding proteins or ribosomal subunits, were excluded (Extended data table 1d).Interestingly, some RBP genes are also enriched in phenotypes such as 'cell death variants' (Extended data table 1e).These genes were retained in the final list for the RNAi screen.The final RBP compendium contained 800 genes (Extended data figure 2B, Extended data table 1f) of which 660 genes are represented in the Ahringer RNAi library (Kamath and Ahringer, 2003;Kamath et al., 2003) (Extended data table 1g).These 660 genes were subjected to the following RNAi screens for activators of egl-1 expression (referred to as 'egl-1 activators').
We first screened the 660 genes for potential egl-1 activators using an egl-1 3′ UTR reporter (Figure 1B, Primary screen) (Sherrard et al., 2017).In this reporter, the egl-1 3′ UTR is fused to a fusion of the coding sequences of gfp and Histone 2B gene his-58 (gfp::his-58), and the expression of the resulting fusion gene is driven by the promoter of the gene mai-2, which is ubiquitously transcribed (Ichikawa et al., 2006).The use of the mai-2 promoter ensures transcription of the reporter in all cells, which allows us to monitor the impact of the 3′ UTR on reporter expression.A single copy of this reporter was inserted into the C. elegans genome, generating the transgene P mai-2 gfp::his-58::egl-1 3′ UTR (bcSi26) (Sherrard et al., 2017).The expression of P mai-2 gfp::his-58::egl-1 3′ UTR is repressed in embryos; however, in oocytes, moderate expression is detected (Figure 1B) (Sherrard et al., 2017).By screening for a decrease in GFP::HIS-58 signal in oocytes, 66 activator candidates were identified (Figure 1A, 1B, Extended data table 2a).After the primary screen, we conducted a secondary (negative) screen for activators that are specific to the egl-1 3′ UTR.To that end, we used a single copy integration of the mai-2 3′ UTR reporter P mai-2 gfp::his-58::mai-2 3′ UTR (bcSi25).This reporter differs from the egl-1 3′ UTR reporter (bcSi26) only in its 3′ UTR but it is ubiquitously expressed in all cells (Figure 1C) (Sherrard et al., 2017).By screening for a decrease in GFP::HIS-58 signal in embryos carrying P mai-2 gfp::his-58::mai-2 3′ UTR (bcSi25), 41 out of 66 candidates were considered general nonspecific activators and were excluded from subsequent analyses.The remaining 25 candidates were considered specific for the egl-1 3′ UTR.The identities of the RNAi clones for these candidates were verified through Sanger sequencing.Twenty of them contained the correct insert (Figure 1A, Extended data table 2b).

Genetic screen by RNA-mediated interference
Genetic screen by RNA-mediated interference (RNAi) was performed using the updated Ahringer RNAi feeding library (Kamath and Ahringer, 2003;Kamath et al., 2003) distributed by Source BioScience Ltd (https://sourcebioscience.com).This library covers ~87% of the currently annotated C. elegans protein-coding genes.Bacterial RNAi clones carrying the constructs that express relevant dsRNAs were cultured in 100 µL of LB medium containing 100 μg/mL carbenicillin in a 96-well plate at 37°C overnight.10 µL of each bacteria culture was seeded into individual wells of a 12-well NGM plate containing 6 mM IPTG and 100 μg/mL carbenicillin as described previously (Rolland et al., 2019).The seeded plates were incubated at 20°C overnight in the dark to induce dsRNA expression before use.
In the primary screen, the egl-1 3′ UTR reporter P mai-2 gfp::his-58::egl-1 3′ UTR (bcSi26) was used to screen for a decrease in gfp::his-58 expression.Ten L3 larvae carrying P mai-2 gfp::his-58::egl-1 3′ UTR (bcSi26) were transferred into each well of the 12-well NGM plate seeded with bacterial RNAi clones.After the animals were fed with bacterial RNAi clones for 48 hours, the expression of gfp::his-58 in nuclei of oocytes was scored.In wild-type animals, this reporter is repressed in embryos but moderately expressed in oocytes and germ cells.If gfp::his-58 expression was reduced in oocytes after the knockdown of an RBP gene, this RBP was considered an activator candidate of egl-1 expression.In this screen, gfp RNAi and control RNAi were used as the positive control and negative control, respectively.
In the secondary (negative) screen, the mai-2 3′ UTR reporter P mai-2 gfp::his-58::mai-2 3′ UTR (bcSi25) was used to exclude non-specific regulators by screening for a decrease in gfp::his-58 expression in 4-cell stage embryos.Candidates that reduced the expression of the mai-2 3′ UTR reporter after their knockdown were excluded.The identities of bacterial RNAi clones were confirmed by Sanger sequencing of the insert in the RNAi construct.
The percentage of NSMsc survival after RNAi-mediated knockdown of RBP genes was determined in the following way.Three L3 stage animals carrying the NSM reporter P tph-1 gfp::his-24 were transferred to NGM plates seeded with bacterial RNAi clones.After three days, L3/L4 stage F1 progenies were scored for extra NSM-like cells, which are found in the anterior pharynx and labelled by the reporter P tph-1 gfp::his-24 (Yan et al., 2013).For RBP genes that cause larval arrest upon knockdown, L1/L2 stage F1 progeny was scored.In this screen, the control RNAi clone was used as a negative control.